Poly-3-hydroxyalkanoic acids (hereinafter sometimes referred to collectively as PHA) are the thermoplastic polyesters which are elaborated and accumulated as energy storage substances by a variety of microorganisms and have biodegradability. In these days waste plastics are disposed of by incineration or burial but these methods of disposal are causative of global warming and ground loosening of reclaimed lands, among other disadvantages. Therefore, with the growing public awareness of the importance of plastics recycling, ways and means for systematized recycling are being explored. However, uses amenable to such recycling are limited and actually the disposal load of waste plastics cannot be completely liquidated by said incineration, burial, and recycling but rather a large proportion of the disposal load is not disposed of but simply left exposed to the elements. There is accordingly a mounting interest in PHA and other biodegradable plastics which, after disposal, would be incorporated into the natural cycle of matter and degradation products of which would not exert ecologically deleterious influences, and their practical utilization are highly desired. Particularly the PHA which microorganisms elaborate and accumulate in their cells is taken up in the carbon cycle of the natural kingdom and it is, therefore, predicted that it will not have any appreciable adverse effects on the ecosystem. In the field of medical treatment, too, it is considered possible to use PHA as an implant material which does not require recovery or a vehicle for drug delivery.
Since the PHA elaborated by microorganisms usually form granules and is accumulated intracellularly, exploitation of PHA as a plastic requires a procedure for separating it from microbial cells. The known technology for the separation and purification of PHA from microbial cells can be roughly classified into the technology which comprises extracting PHA from the cells with an organic solvent in which PHA is soluble and the technology which comprises removing the cell components other than PHA by cell disruption or solubilization.
Referring to the separation and purification technology of PHA involving extraction with an organic solvent, the extraction technique utilizing a halogen-containing hydrocarbon, such as 1,2-dichloroethane or chloroform, as the solvent in which PHA is,soluble is known (Japanese Kokai Publication Sho-55-118394, Japanese Kokai Publication Sho-57-65193). However, since these halogen-containing hydrocarbons are hydrophobic solvents, a pre-extraction procedure, such as drying the cells in advance or otherwise, allowing the solvent to directly contact the intracellular PHA is required. Moreover, in such a technology, dissolving PHA at a practically useful concentration (for example, 5%) or higher gives only an extract which is so highly viscous that it involves considerable difficulties in separating the undissolved residues of microbial cells from the PHA-containing solvent layer. Furthermore, in order that PHA may be reprecipitated from the solvent layer at a high recovery, some PHA-insoluble solvent, such as methanol or hexane, need to be used in a large quantity, e.g. 4 to 5 volumes based on the solvent layer, and thus a vessel of large capacity is required for reprecipitation. In addition, the necessary quantity of solvents is so large that both the solvent recovery cost and the cost of lost solvents are enormous. Furthermore since the use of organohalogen compounds tends to be limited these days for protection of the environment, industrial application of this technology has many obstacles to surmount.
Under the circumstances, there has been proposed an extraction technology using a solvent which is not only capable of dissolving PHA but also miscible with water, for example a hydrophilic solvent such as dioxane (Japanese Kokai Publication Sho-63-198991), propanediol (Japanese Kokai Publication Hei-02-69187), or tetrahydrofuran (Japanese Kokai Publication Hei-07-79788). These methods appear to be favorable partly because PHA can be extracted not only from dry cells but also from wet cells and partly because precipitates of PHA can be obtained by mere cooling of the solvent layer separated from the microbial cell residues. However, even with these methods, the problem of high viscosity of the PHA-containing solvent layer remains to be solved. In addition, while heating is required for enhancing the extraction efficiency, the heating in the presence of water unavoidably results in a decrease in molecular weight due to hydrolysis of PHA and a poor recovery of PHA.
On the other hand, as the technology of removing the cell components other than PHA by solubilization for separation of PHA, J. Gen. Microbiology, 19, 198-209 (1958) describes a technology which comprises treating a suspension of microbial cells with sodium hypochlorite to solubilize cell components other than PHA and recovering PHA. This technology is simple process-wise but the necessity to use a large amount of sodium hypochlorite is a factor leading to a high production cost. Moreover, in view of the marked decrease in molecular weight of PHA and the appreciable amount of chlorine left behind in PHA, this technology is not considered to be suitable for practical use. Japanese Kokoku Publication Hei-04-61638 describes a process for separating PHA which comprises subjecting a suspension of PHA-containing microbial cells to a heat treatment at a temperature of 100° C. or higher to disrupt the cellular architecture and, then, subjecting the disrupted cells to a combination treatment with a protease and either a phospholipase or hydrogen peroxide to solubilize the cell components other than PHA. This technology is disadvantageous in that because the heat treatment induces denaturation and insolubilization of the protein, the load of subsequent protease treatment is increased and that the process involves many steps and is complicated.
As a technology for disrupting PHA-containing microbial cells, there also has been proposed a method which comprises treating microbial cells with a surfactant, decomposing the nucleic acids released from the cells with hydrogen peroxide, and separating PHA (Japanese Kohyo Publication Hei-08-502415) but the waste liquor containing the surfactant develops a copious foam and, in addition, has a high BOD load value. From these points of view, the use of a surfactant is objectionable for production on a commercial scale.
There has also been proposed a technology for separating PHA which comprises disrupting PHA-containing microbial cells with a high-pressure homogenizer (Japanese Kokai Publication Hei-07-177894 and Japanese Kokai Publication Hei-07-31488). However, this technology has the drawback that although a suspension of microbial cells is subjected to a high-pressure treatment at least 3 times, or 10 times at elevated temperature depending on cases, the purity of PHA that can be attained is as low as about 65 to 89%. There has also been proposed a technology for separating PHA which comprises adding an alkali to a suspension of PHA-containing microbial cells, heating the suspension, and disrupting the cells (Japanese Kokai Publication Hei-07-31487). However, this technology is disadvantageous in that the purity of the product polymer that can be attained is as low as 75.1 to 80.5% and that if the level of addition of the alkali be raised to improve the yield, the molecular weight of the polymer would be decreased. Several techniques for carrying out physical disruption after addition of an alkali have been proposed (Bioseparation, 2, 95-105, 1991, Japanese Kokai Publication Hei-07-31489) but since the alkali treatment alone results in the extracellular release of only a small amount of cell components and some of such cell components are retained in the PHA fraction even after subsequent high-pressure disruption treatment, these techniques are invariably inefficient. Thus, PHA of high purity cannot be separated unless the microbial cell suspension is subjected to at least 5 cycles of high-pressure treatment and even then the purity of PHA is as low as about 77 to 85%. The technologoy involving addition of an alkali has an additional drawback; generally the cell components released from microbial cells, particularly nucleic acids, increase the viscosity of the cell suspension to make subsequent processing difficult.
There has also been proposed a technology in which a suspension of PHA-containing microbial cells is adjusted to an acidity lower than pH 2 and PHA is separated at a temperature not below 50° C. (Japanese Kokai Publication Hei-11-266891). However, this technology is disadvantageous in that the treatment under the strongly acidic condition below pH 2 is undesirable for production on a commercial scale, that the acid treatment must be followed by adjustment to the alkaline side for enhanced purity but this entails massive salt formation, and that the molecular weight of the product PHA is decreased from 2,470,000 to about 1,000,000.